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Towards Myosin Powered Lab-on-a-Chip Devices
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. (Alf Månsson)ORCID iD: 0000-0001-6878-3142
2013 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Mot utvecklandet av myosindrivna laboratorier på chip (Swedish)
Abstract [en]

Myosins are protein motors that use chemical energy in the form of adenosinetriphosphate to produce force and motion. These molecular motors might be usedto power transportation in Lab-on-a-chip devices where a series of laboratory tasks(e.g. separation, concentration and detection) are performed in one sequence on asmall chip. Because of the small size, lab-on-a-chip devices are predicted to befaster and more sensitive than conventional systems. Further potential advantagesinclude cost efficiency and the possibility to perform many analyzes in parallel.Substituting microfluidics with myosin based transport would allow furtherminiaturization and make lab-on-a-chip devices more readily portable by reducingthe need for external power supplies. However, there are also limitations thathamper the development of such devices. Here we investigate several aspects of amyosin powered lab-on-a-chip device and present ways to overcome criticallimitations. First we demonstrate covalent attachment of antibodies to actinfilament shuttles with retained ability of the filaments to be propelled by myosinfragments, previously believed to be difficult. Secondly we develop a separationmethod to overcome the deleterious effects of body fluids on the actomyosinsystem. Thirdly, we explore the possibility to concentrate actin shuttles on ananostructured surface and achieve >20 times concentration in <1 min. Monte-Carlo simulations of the concentration process suggest further room forimprovement. Fourth, we develop novel techniques for fast and automaticdetection of fluorescence at certain check points which improves S/N ratio >20times. Finally, we take the first steps towards the development of threedimensional,nanowire-based transport systems, important both for lab-on-a-chipapplications and fundamental studies. Our results demonstrate the potential of amyosin based lab-on-a-chip device and lay the foundation for furtherdevelopments. Thus, we anticipate that this work will influence future studiestowards a complete diagnostic lab-on-a-chip work-up based on molecular motors.In addition, the work might also have implications for the development of futurebiocomputation and drug screening devices as well as novel biophysical studies ofthe actomyosin system.

Place, publisher, year, edition, pages
Växjö: Linnaeus University Press, 2013. , 200 p.
Series
Linnaeus University Dissertations, 144/2013
Keyword [en]
myosin, actin, molecular motors, lab-on-a-chip, nanobiotechnology, bionanotechnology, diagnostics, nanowires, nanowire arrays
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biomedical Sciences
Identifiers
URN: urn:nbn:se:lnu:diva-28384ISBN: 978-91-87427-45-9 (print)OAI: oai:DiVA.org:lnu-28384DiVA: diva2:642612
Public defence
2013-09-13, N2007, Västergård, Smålandsgatan 26E, Kalmar, 09:00 (English)
Opponent
Supervisors
Funder
EU, FP7, Seventh Framework Programme, 228971
Available from: 2013-09-10 Created: 2013-08-22 Last updated: 2016-05-03Bibliographically approved
List of papers
1. Tracking Actomyosin at Fluorescence Check Points
Open this publication in new window or tab >>Tracking Actomyosin at Fluorescence Check Points
2013 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 3, 1092Article in journal (Refereed) Published
Abstract [en]

Emerging concepts for on-chip biotechnologies aim to replace microfluidic flow by active, molecular-motor driven transport of cytoskeletal filaments, including applications in bio-simulation, biocomputation, diagnostics, and drug screening. Many of these applications require reliable detection, with minimal data acquisition, of filaments at many, local checkpoints in a device consisting of a potentially complex network of channels that guide filament motion. Here we develop such a detection system using actomyosin motility. Detection points consist of pairs of gold lines running perpendicular to nanochannels that guide motion of fluorescent actin filaments. Fluorescence interference contrast (FLIC) is used to locally enhance the signal at the gold lines. A cross-correlation method is used to suppress errors, allowing reliable detection of single or multiple filaments. Optimal device design parameters are discussed. The results open for automatic read-out of filament count and velocity in high-throughput motility assays, helping establish the viability of active, motor-driven on-chip applications.

National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-24539 (URN)10.1038/srep01092 (DOI)000313885400004 ()2-s2.0-84873142047 (Scopus ID)
Available from: 2013-02-25 Created: 2013-02-25 Last updated: 2017-12-06Bibliographically approved
2. Ultrafast molecular motor driven nanoseparation and biosensing
Open this publication in new window or tab >>Ultrafast molecular motor driven nanoseparation and biosensing
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2013 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 48, 145-152 p.Article in journal (Refereed) Published
Abstract [en]

Portable biosensor systems would benefit from reduced dependency on external power supplies as well as from further miniaturization and increased detection rate. Systems built around self-propelled biological molecular motors and cytoskeletal filaments hold significant promise in these regards as they are built from nanoscale components that enable nanoseparation independent of fluidic pumping. Previously reported microtubule-kinesin based devices are slow, however, compared to several existing biosensor systems. Here we demonstrate that this speed limitation can be overcome by using the faster actomyosin motor system. Moreover, due to lower flexural rigidity of the actin filaments, smaller features can be achieved compared to microtubule-based systems, enabling further miniaturization. Using a device designed through optimization by Monte Carlo simulations, we demonstrate extensive myosin driven enrichment of actin filaments on a detector area of less than 10 μm2, with a concentration half-time of approximately 40 s. We also show accumulation of model analyte (streptavidin at nanomolar concentration in nanoliter effective volume) detecting increased fluorescence intensity within seconds after initiation of motor-driven transportation from capture regions. We discuss further optimizations of the system and incorporation into a complete biosensing workflow.

Place, publisher, year, edition, pages
Elsevier, 2013
Keyword
Actin filament; Diagnostics; Electron beam lithography; Heavy meromyosin; Nanoseparation; Monte-Carlo simulation
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-28382 (URN)10.1016/j.bios.2013.03.071 (DOI)000321085600024 ()2-s2.0-84877888579 (Scopus ID)
Funder
EU, FP7, Seventh Framework Programme, 228971The nanometer Structure Consortium at Lund UniversitySwedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2013-08-22 Created: 2013-08-22 Last updated: 2017-12-06Bibliographically approved
3. Antibodies Covalently Immobilized on Actin Filaments for Fast Myosin Driven Analyte Transport
Open this publication in new window or tab >>Antibodies Covalently Immobilized on Actin Filaments for Fast Myosin Driven Analyte Transport
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 10, e46298Article in journal (Refereed) Published
Abstract [en]

Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 mu m(-1)). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments.

National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-22698 (URN)10.1371/journal.pone.0046298 (DOI)000309454000032 ()2-s2.0-84867081685 (Scopus ID)
Available from: 2012-12-05 Created: 2012-12-05 Last updated: 2017-12-07Bibliographically approved
4. Magnetic capture from blood rescues molecular motor function in diagnostic nanodevices
Open this publication in new window or tab >>Magnetic capture from blood rescues molecular motor function in diagnostic nanodevices
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2013 (English)In: Journal of Nanobiotechnology, ISSN 1477-3155, E-ISSN 1477-3155, Vol. 11, 14Article in journal (Refereed) Published
Abstract [en]

Background: Introduction of effective point-of-care devices for use in medical diagnostics is part of strategies to combat accelerating health-care costs. Molecular motor driven nanodevices have unique potentials in this regard due to unprecedented level of miniaturization and independence of external pumps. However motor function has been found to be inhibited by body fluids. Results: We report here that a unique procedure, combining separation steps that rely on antibody-antigen interactions, magnetic forces applied to magnetic nanoparticles (MPs) and the specificity of the actomyosin bond, can circumvent the deleterious effects of body fluids (e.g. blood serum). The procedure encompasses the following steps: (i) capture of analyte molecules from serum by MP-antibody conjugates, (ii) pelleting of MP-antibody-analyte complexes, using a magnetic field, followed by exchange of serum for optimized biological buffer, (iii) mixing of MP-antibody-analyte complexes with actin filaments conjugated with same polyclonal antibodies as the magnetic nanoparticles. This causes complex formation: MP-antibody-analyte-antibody-actin, and magnetic separation is used to enrich the complexes. Finally (iv) the complexes are introduced into a nanodevice for specific binding via actin filaments to surface adsorbed molecular motors (heavy meromyosin). The number of actin filaments bound to the motors in the latter step was significantly increased above the control value if protein analyte (50-60 nM) was present in serum (in step i) suggesting appreciable formation and enrichment of the MP-antibody-analyte-antibody-actin complexes. Furthermore, addition of ATP demonstrated maintained heavy meromyosin driven propulsion of actin filaments showing that the serum induced inhibition was alleviated. Detailed analysis of the procedure i-iv, using fluorescence microscopy and spectroscopy identified main targets for future optimization. Conclusion: The results demonstrate a promising approach for capturing analytes from serum for subsequent motor driven separation/detection. Indeed, the observed increase in actin filament number, in itself, signals the presence of analyte at clinically relevant nM concentration without the need for further motor driven concentration. Our analysis suggests that exchange of polyclonal for monoclonal antibodies would be a critical improvement, opening for a first clinically useful molecular motor driven lab-on-a-chip device.

Keyword
Magnetic nanoparticle, Biomolecular motor, Myosin, Nanoseparation, Lab-on-a-chip, Bioconjugation
National Category
Medical Biotechnology
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-27565 (URN)10.1186/1477-3155-11-14 (DOI)000319316500001 ()2-s2.0-84877003874 (Scopus ID)
Available from: 2013-07-17 Created: 2013-07-17 Last updated: 2017-12-06Bibliographically approved

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